This proposal describes a lineage marking strategy that is designed to discriminate antigen-experienced T cells from their naive precursors. The key feature of the strategy is the synchronization of a stable genomic alteration with the activation of T cells during immune responses. We will accomplish this synchronization by using the transcriptional control elements of the mouse OX40 locus to drive expression of the Cre recombinase gene. OX40 is a gene that is only expressed in activated T cells, thus Cre expression should be confined to this population of cells. The target for the Cre recombinase will be a novel reporter cassette containing two cistrons. The promoter-proximal cistron will be expressed constitutively in naive T cells and will therefore serve as a marker for these cells. The cistron for this upstream reporter will be flanked by LoxP sites (recognition sites for the Cre recombinase) and will encode a protein that can be readily detectable by flow cytometry or other facile procedures. Cre-mediated recombination during T cell activation will then delete this cistron thereby allowing for expression of a downstream cistron encoding a different reporter molecule. This second reporter will then be used to identify post-activation or antigen-experienced T cells, a population that should include memory T cells. We will use this lineage marking strategy to characterize the immunologic properties of antigen- experienced T cells and to compare then to those of naive precursor cells. Properties such as lifespan, proliferative cycle and sensitivity to activating stimuli will be examined. The development of this marking scheme will expand the repertoire of tools available for the identification of subpopulations of T lymphocytes. Moreover, this study will provide important information about the reactivity and homeostatic activity of antigen-experienced T cells. This approach has general relevance to the issue of how immune responses are regulated and may be of particular value to the study of autoimmune diseases. Such diseases generally depend on chronic autoreactivity of T cells and may arise as a consequence of inadequate immunologic regulation by the antigen- experienced T cells which are the subject of this proposal.